The protective effects of humoral immunity are known to be mediated by a family of structurally related glycoproteins called antibodies. Antibodies initiate their biological activity by binding to antigens. Antibody binding to antigens, which may be covalent or non-covalent, is exquisitely specific for one antigen and is often very strong.
Antibodies are produced in a membrane-bound form by B-lymphocytes. Blood contains many different antibodies, each derived from a clone of B-cells and each having a distinct structure and specificity for antigen. Antibodies are present in the cytoplasmic compartment and on the surface of B-lymphocytes, in the plasma, in interstitial fluid of the tissues and in secretory fluids such as saliva and mucous. Surfaces of immunoeffector cells, such as mononuclear phagocytes, natural killer cells and mast cells also have antibodies.
All antibodies are similar in their overall structure, accounting for certain similarities in physiochemical features such as charge and solubility. All antibodies have a common core structure of two identical light chains, each about 24 kilodaltons, and two identical heavy chains of about 55-70 kilodaltons each. One light chain is attached to each heavy chain, and the two heavy chains are attached to each other. Both the light and heavy chains contain a series of repeating homologous units, each of about 110 amino acid residues in length which fold independently in a common globular motif, called an immunoglobulin (Ig) domain. The region of an antibody formed by the association of the two heavy chains is hydrophobic. Antibodies, and especially monoclonal antibodies, are known to cleave at the site where the light chain attaches to the heavy chain when they are subjected to adverse physical or chemical conditions. Because antibodies contain numerous cysteine residues, they have many cysteine-cysteine disulfide bonds. All Ig domains contain two layers of beta-pleated sheets with three or four strands of anti-parallel polypeptide chains.
Despite their overall similarity, antibody molecules can be divided into a small number of distinct classes and subclasses based on physiochemical characteristics such as size, charge and solubility, and on their behavior in binding to antigens. In humans, the classes of antibody molecules are: IgA, IgD, IgE, IgG and IgM. Members of each class are said to be of the same isotype. IgA and IgG isotypes are further subdivided into subtypes called IgA.sub.1, IgA.sub.2 and IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4. The heavy chains of all antibodies in an isotype share extensive regions of amino acid sequence identity, but differ from antibodies belonging to other isotypes or subtypes. Heavy chains are designated by the letters of the greek alphabet corresponding to the overall isotype of the antibody, e.g., IgA contains .alpha., IgD contains .delta., IgE contains .epsilon., IgG contains .gamma., and IgM contains .mu. heavy chains. IgG, IgE and IgD circulate as monomers, whereas secreted forms of IgA and IgM are dimers and pentamers, respectively, stabilized by the J chain. Some IgA molecules exist as trimers.
There are 1.times.10.sup.7, and perhaps as many at 10.sup.9, structurally different antibody molecules in every individual, each with the unique amino acid sequence in their antigen combining sites. Sequence diversity in antibodies is confined to three short stretches within the amino terminal domains of the heavy and light chains. The amino acid sequences of the amino terminal domains are called variable (V) regions, to distinguish them from the more conserved constant (C) regions.
Antibodies have several known therapeutic applications. For example, they may be used to bind to and block cell markers and receptors. Antibodies to microorganisms may be used to inhibit or inactivate the microorganism and/or prevent or treat disease conditions caused by these microorganisms. When antibodies bind to microorganisms, they enhance their recognition and destruction by macrophages. Antibodies may be used as anticytokines, antichemokines, antihormones, antiinflammatories and immunosuppressors or as antineutrophil adhesion agents. Antibodies, especially monoclonal antibodies, may be used systemically to deliver therapy. In these cases antibodies are often used as immunoconjugates, immunoliposomes or immunomicrospheres. Antibodies can be used as apoptosis stimulators and as recognizers of cancerous and precancerous cells. Known examples of antibodies which may be useful therapeutically include the following.
MedImmune Inc. is studying the use of humanized anti-RSV monoclonal antibodies and markets a polyclonal anti-RSV antibody from donor blood (RespiGam) to treat respiratory syncytial virus (RSV) infections. MedImmune also markets CytoGam, an anti-CMV (cytomegalovirus) human immune globulin for the treatment of CMV infection. IDEC and Genentech are jointly performing clinical trials of a chimeric mouse-human monoclonal antibody (rituximab) aimed at the CD20 antigen found on mature B cells and most non-Hodgkin's lymphoma tumors for use in treating relapsed or refractory low-grade non-Hodgkin's lymphoma. GalaGen is studying the use of the polyclonal antibody Diffistat-G for treatment of Clostridium difficile antibiotic associated diarrhea. Smith Kline and Schering Plough are developing an anti-IL-5 antibody which has been shown in clinical trials to prevent eosinophilic inflammation and airway constriction. An anti-IgE antibody is being developed by Genentech to "switch-off" allergies. Monoclonal antibody Rhu-Mab-E25, which is a humanized chimeric IgG.sub.1 monoclonal antibody for a unique epitope on human high affinity IgE receptors (FceRI), has been shown to reduce free IgE levels after the first administration by injection. It attenuated both early and late phase responses to inhaled allergens after multiple injections. Examples of antibodies used therapeutically also include a nebulized IgG (Sandoz), which is used intranasally against respiratory syncytial virus (RSV); HNK20 (Oravax), an anti-RSV IgA; and 4B9 (Bristol Myers-Squibb), an anti-group B Streptococcus IgM monoclonal antibody. Other therapeutically useful antibodies include anti-CD4 antibodies, anti-IL-2 antibodies and anti-IL-4 antibodies.
The immunotherapy of respiratory syncytial virus infection using small particle aerosols of IgG has been disclosed by Piazza et al. (The Journal of Infectious Diseases, Vol. 166, pp. 1422-1424, 1992) In this study it was shown that a 15-minute exposure to an aerosolized 5% solution of IgG effected a 50-fold reduction in pulmonary virus. Brown (Aerosol Science and Technology, Vol. 24, pp. 45-56, 1996) discloses the use of antibodies as inhibitors or antagonists of cytokines to depress respiratory inflammatory diseases or allergen-induced asthmatic responses. Also disclosed is local respiratory delivery of pathogen-specific antibody for treatment of acute viral or bacterial respiratory infections. Antibody liposomes, i.e., immunoliposomes, are disclosed by Maruyama et al. in Biochemica et Biophysica Acta, Vol. 1234, pp. 74-80, 1995. Coating liposomes with antibody leads to enhanced uptake of the immunoliposome by the reticuloendothelial system. Human monoclonal antibodies are known to be useful as antitumor agents. A mouse/human monoclonal IgG, antibody specific for the Lewis Y antigen found on the surface of tumor cells is disclosed by Paborji et al. (Pharmaceutical Research, Vol. 11, No. 5, pp. 764-771, 1994). The use of antibodies in metered-dose propellant driven aerosols for passive antibody aerosol therapy against respiratory infections is disclosed in Brown et al. (Journal of Immunological Methods, Vol. 176, pp. 203-212, 1994). Immune responses in the respiratory tract are of great importance for protection against infections of the respiratory system and for their involvement in respiratory allergies and asthma. Effective targeting of immunomodulating reagents including antibodies to the respiratory tract is shown to be of benefit in increasing local immunity to respiratory pathogens or decreasing immune mediated respiratory pathology. Inhaled immunoconjugates, immunoliposomes or immunomicrospheres have application in the lung as killers of cancer cells (immunoconjugates) or, in the case of immunoliposomes and microspheres, as stealth delivery particles of a variety of therapeutic agents. An IgM anti-group B Streptococcus monoclonal antibody is disclosed by Gombotz et al. (Pharmaceutical Research, Vol. 11, pp. 624-632, 1994).
Over the years certain drugs have been sold in compositions suitable for forming a drug dispersion for oral inhalation (pulmonary delivery) to treat various conditions in humans. Such pulmonary drug delivery compositions are designed to be delivered by inhalation by the patient of the drug dispersion so that the active drug within the dispersion can reach the lung. It has been found that certain drugs delivered to the lung are readily absorbed by the alveolar region directly into the blood circulation. Pulmonary delivery is particular promising for delivery of macromolecules such as proteins, polypeptides, high molecular weight polysaccharides and nucleic acids, which are difficult to deliver by other routes of administration. Such pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
Pulmonary drug delivery can itself be achieved by different approaches, including liquid nebulizers, aerosol-based metered-dose inhalers (MDI's) and dry powdered dispersion devices. Chlorofluorocarbon (CFC) based MDI's are losing favor because of their adverse effect on the environment. Dry powder dispersion devices, which do not rely on CFC aerosol technology, are promising for delivering drugs that may be readily formulated as dry powders. Many otherwise labile macromolecules may be stably stored as lyophilized or spray dried powders, either by themselves or in combination with suitable powder carriers.
The ability to deliver pharmaceutical compositions as dry powders, however, is problematic in certain respects. The dosage of many pharmaceutical compositions is often critical, so it is desirable that dry powder delivery system be able to accurately, precisely and reliably deliver the intended amount of drug. Moreover, many pharmaceutical compositions, including antibodies, are quite expensive. Thus, the ability to efficiently formulate, process, package and deliver the dry powders with minimal loss of drug is critical. While the permeability of natural macromolecules in the lung is well known, combined inefficiency of macromolecule production processes and macromolecule delivery has limited commercialization of dry macromolecule powders for pulmonary delivery. It is also essential that dry powders for pulmonary delivery be readily dispersible prior to inhalation by the patient in order to assure adequate distribution and systemic absorption.
An important requirement for hand held and other powder delivery devices is efficiency. It is important that the delivered dose be relatively high to reduce the number of breaths required to achieve a total dosage. The ability to achieve both adequate dispersion and small dispersed volumes is a significant technical challenge that requires in part that each unit dosage of the powder composition be readily and reliably dispersible. Certain pulmonary delivery devices, such as those disclosed in U.S. Pat. No. 5,458,135 and International Patent Publication WO96/09085 are useful for pulmonary delivery of dry powder drugs.
Spray drying is a conventional chemical processing operation used to produce dry particulate solids from a variety of liquid and slurry starting materials. The use of spray drying for the formulation of dry powder pharmaceuticals is known but has usually been limited to small molecule and other stable drugs which are less sensitive to thermal degradation and other rigorous treatment conditions. The use of spray drying for the preparation of biological macromolecule compositions, including antibodies, can be problematic since such macromolecules are often labile and subject to degradation when exposed to high temperatures and other aspects of the spray drying process. Excessive degradation of the macromolecules can lead to drug formulations lacking in the requisite purity.
It can also be difficult to control particle size and particle size distribution in compositions produced by spray drying. For pulmonary delivery it is critical that the average particle size be maintained in a respirable range and that the amount of the composition comprising particles outside the target size range be minimized. Moreover, it can sometimes be difficult to achieve a desired low moisture content required for physical and chemical stability in the final particulate product, particularly in an economic manner. Finally, and perhaps most importantly, it has been difficult to produce the small particles necessary for pulmonary delivery in an efficient manner. For high value macromolecular drugs, high collection efficiencies, i.e., the amount of particulate drug recovered from the process in a usable form, are important. While spray drying has been used to prepare powders of macromolecules in laboratory scale equipment, commercial spray dryers are not designed to produce powders in the pulmonary size range. The methods for atomization, drying powder and collection must be modified to economically produce a protein powder with the desired product characteristics for pulmonary delivery and in sufficient yield and at commercially acceptable production rates, i.e., in excess of 30 grams per hour. Useful methods are disclosed, for example, in International Patent Application No. PCT/US97/07779, the disclosure of which is incorporated herein by reference in its entirety.